Numerous Gram-negative bacteria including many NIAID Biodefense priority pathogens utilize type III secretion systems (T3SSs) to deliver tens of proteins into host cells. While many of the components of these complex machines are highly conserved and functionally interchangeable, each bacterium delivers its own unique set of effectors into host cells. A complete understanding of how the bacteria ensure that only small subsets of the thousands of proteins they encode are specifically delivered into host cells remains unclear. My laboratory recently developed a novel yeast-based visualization assay to identify interacting proteins in living cells, the Protein Interaction Platform assay or PIP. Remarkably, we found that one chaperone, Spa15, interacted with 10 effectors, nine of which we subsequently established require Spa15 for their efficient type III secretion. Furthermore, we find that the Spa15 homologs from eight bacterial species are functionally interchangeable with Spa15. All nine proteins recognize a defined chaperone-binding domain sequence. Given the functional interchangeable of these eight chaperones, we hypothesize that they recognize effectors and deliver effectors to the secretion apparatus by conserved mechanisms. The goals of the proposal are to begin to determine the molecular mechanisms underlying these important steps in type III secretion. Specifically we propose to (a) investigate how class IB chaperones differentiate between effectors and housekeeping proteins (Aim 1) and (b) determine how chaperone/effector complexes are recruited to the type III secretion apparatus (Aim 2). Insights gained from these studies will likely result in generating new directions for the development of novel agents for the treatment of a variety of diseases associated with Gram-negative bacterial infections.